Mucosal administration of substances to mammals

ABSTRACT

A novel method for the mucosal administration of a substance to a mammal is provided. The method comprises contacting a mucosal surface of the mammal with the substance in combination with a Biovector. The Biovector has a core that comprises a natural polymer, or a derivative or a hydrolysate of a natural polymer, or a mixture thereof. A preferred natural polymer is a polysaccharide or an oligosaccharide. The core is optionally coated with an amphiphilic compound, such as a lipid.

BACKGROUND OF THE INVENTION

A large number of pharmaceutical substances for various purposes havebeen developed for introduction into animals, including humans. Thesesubstances include therapeutic agents, such as drugs; prophylacticagents, such as antigens for use in vaccines; and diagnostic agents,such as labeled imaging agents. These substances may be introduced by avariety of enteral and parenteral modes of administration.

There has recently been a proliferation of potential and realizedpharmaceutical compounds that are macromolecules, such as proteins andnucleic acid molecules. These macromolecular compounds presentparticular problems for drug delivery, since they tend to be unstable,poorly absorbed, and easily metabolized.

There has also been renewed interest in the mucosal administration ofpharmaceutical substances. The mucosa refers to the epithelial tissuethat lines the internal cavities of the body, such as thegastrointestinal tract, the respiratory tract, the lungs, and thegenitalia. For the purpose of this specification, the mucosa will alsoinclude the external surface of the eye, i.e. the cornea.

Some common modes of mucosal administration include oral and nasaladministrations. Currently known methods of ocular administration aresubject to several limitations that compromise their effectiveness.These problems include rapid nasolacrimal drainage, poor cornealpenetration, non-productive conjunctival loss, and unwanted systemicexposure.

Almeida et al. have reviewed the mucosal administration of vaccines ingeneral, and nasal administration of vaccines in particular in theJournal of Drug Targeting 3, 456-467 (1996). Mucosal immunity is basedon the existence in the mucosa of mucosal-associated lymphoid tissue(MALT). These include gut-associated lymphoid tissue (GALT),bronchus-associated lymphoid tissue (BALT), and nasal-associatedlymphoid tissue (NALT). Mucosal immunization is capable of inducing botha local (IgA) and systemic (IgG) immune response. In addition, there isa common mucosal immune system, whereby an antigen enters the MALT at alocal site, and is transported through the regional lymph nodes to othermucosal surfaces, where an immune response is also induced.

Pharmaceutical substances may be administered either in the absence orin the presence of a carrier. Various purposes may be served by suchcarriers, such as the controlled release of biologically activemolecules, and the targeting of biologically active molecules tospecific tissues.

Illum et al. investigated three microspheres as potential nasal drugdelivery systems. The microspheres were albumin, starch, andDEAE-Sephadex. Although these microspheres showed some promise, certainproblems still need to be overcome.

For example, Illum et al. reported that the size of the microspheresmust be greater than 10 μm. Such large particles, however, have certaindisadvantages. For example, they cannot be sterilized byultrafiltration, requiring other methods, such as the use ofpreservatives. In addition, Illum et al. reported difficulty releasingdrugs from microspheres having a cationic charge. There are advantagesto positively charged microspheres, and the problems reported by Illumet al. must be overcome.

Liposomes are often used as carriers for substances. They have shownpotential as controlled release drug delivery systems and asimmunological adjuvants. The use of liposomes as carriers for vaccinesis discussed in the article by Almeida et al. mentioned above. Morespecifically, the use of liposomes as carriers for influenza vaccineswas discussed by El Guink et al., Vaccine 7, 147-151 (1989), and in U.S.Pat. No. 4,196,191 of the Burroughs Wellcome Company and InternationalPCT Application WO 92/03162 of the Wellcome Foundation.

There are, however, disadvantages in the use of liposomes as carrier foractive compounds. For example, only small amounts of one compound cangenerally be incorporated in a liposome, and the ratio of activecompound to lipid is low. Moreover, the active compound is oftenreleased too early.

Liposomes also present certain manufacturing disadvantages. For example,detergents and solvents are used to increase solubility during one phaseof the manufacturing process. These detergents and solvents must beeliminated from the drug at a later stage.

Other difficulties in using liposomes as drug delivery systems have beenreported by Meisner in Chapter 3, page 31 of Pharmaceutical ParticulateCarriers--Therapeutic Applications, A. Roland, ed., Marcel Dekker, 1993.There is, therefore, the need for a more flexible carrier forsubstances.

Other carriers for substances have been described in U.S. Pat. No.4,921,757 and 4,900,556 of the Massachussets Institute of Technology;U.S. Pat. No. 5,354,853 of Genzyme Corporation; and European Patent 352295 of Access Pharmaceuticals, Inc. For example, the Access patentdescribes a carrier for drugs and diagnostic agents having a multivalentbinding agent, such as heparin. The multivalent binding agent isspecific for endothelial surface determinants, and may be as large asthree micrometers.

The carriers described in the Access patent have, however certaindisadvantages. First, the Access carriers bind specifically toendothelial cells. Also, the Access patent describes only carrierspre-loaded with the drug or diagnostic agent prior to administration.Such methods can lead to instability. Thus, Examples X and XII on page19 of the Access patent measure stability in hours. Also, the carriersdescribed in the Access patent are generally too large to be subjectedto microfiltration.

In addition to those mentioned above, numerous other microspheres andnanospheres are known. These include polyacrylate, latex, andpolylactide polymers. Bjork and Edman, International Journal ofPharmaceutics 47, 233 (1988) reported that starch, cellulose, anddextran microspheres can act as absorbtion enhancers if they satisfycertain criteria, i.e., they must be water absorbtive, water insoluble,and administered in powder form to the nose.

A new type of improved carrier was described by Biovector Therapeutics,S.A. in International PCT Application WO 94/20078. These carriers,called Supramolecular Biovectors (SMBVs) act as solvated suspensions inwater, while still maintaining their integrity assubstance-encapsulating particles. These SMBVs comprise a non-liquidhydrophylic core, such as a cross-linked polysaccharide or across-linked oligosaccharide and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid. TheBiovector optionally has cationic or anionic ligands grafted into thepolysaccharide or oligosaccharide core. The Biovector also optionallycontains a layer of lipid compounds grafted onto the core by covalentbonds. See International PCT Application WO 94/23701. These Biovectorshave been described as being useful in vaccines, such as in CMVvaccines. See International PCT Application WO 96/06638.

There is a need for a carrier that is capable of delivering substancesto animals, including humans, efficiently, and that avoids thedisadvantages of prior art carriers. An object of the present inventionis to provide a method for the administration of biologically activemolecules and other substances to mammals in a way that avoids thedisadvantages discussed above. More specifically, an object of thepresent invention is to provide a method for administering substances tomammals by means of a carrier that directs the substance to the mucosain a non-specific manner, that is capable of being loaded with thesubstance immediately prior to administration, that is of a sizesusceptible to microfiltration, and that is stable for up to twelvemonths and even one or more years.

SUMMARY OF THE INVENTION

These and other objectives as will be appreciated by those havingordinary skill in the art have been met by providing a novel method forthe mucosal administration of a substance to a mammal. The methodcomprises contacting a mucosal surface of the mammal with the substancein combination with a Biovector. The Biovector has a core that comprisesa natural polymer, or a derivative or a hydrolysate of a naturalpolymer, or a mixture thereof.

The invention further relates to the use of Biovectors associated withone or more biologically active compounds to prepare a composition fortherapeutic or preventative purposes, especially against infectiousagents, via mucosal administration to a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the rate of clearance of ¹⁴ C-radiolabeled Biovectors fromthe nasal mucosa following administration of ¹⁴ C-radiolabeledBiovectors to rats. The percent of the ¹⁴ C radiolabel remaining in thenasal turbinate (cavity) is plotted against the number of hoursfollowing administration. The protocol is described in Example II. Thesquares represent cationic Biovectors, the diamonds represent anionicBiovectors, and the triangles represent free ¹⁴ C (control).

FIG. 2 shows the concentration in ng/ml of the radiolabel found in theplasma three, six, and twelve hours following administration of ¹⁴C-radiolabeled Biovectors to rats in accordance with the protocoldescribed in Example II. The filled triangles represent SMBV-P1, thefilled circles represent SMBV-P2, the filled squares represent SMBV-P3,the empty triangles represent SMBV-Q1, the empty circles representSMBV-Q2, and the empty squares represent SMBV-Q3.

DETAILED DESCRIPTION OF THE INVENTION

In the description of the invention below, the following interpretationswill apply. The word "comprise" followed by an element of the inventionused in describing an embodiment of the invention means that theembodiment includes, but is not necessarily limited to, that element.The embodiment may include other members of the same element or otherelements as well. An element disclosed in the singular, i.e."substance," does not preclude the presence of more than one element,i.e. "substances." All numbers are approximate, unless the language ofthe specification or its context indicates otherwise.

It has unexpectedly been discovered that Biovectors, as described inInternational PCT Application WO 94/23701, WO 94/20078, and WO 96/06638,are particularly well suited for the mucosal administration ofsubstances to mammals, including farm animals, pet animals, laboratoryanimals, and humans. The mucosa refers to the epithelial tissue thatlines the internal cavities of the body. For example, the mucosacomprises the alimentary canal, including the mouth, esophagus, stomach,intestines, and anus; the respiratory tract, including the nasalpassages, trachea, bronchi, and lungs; and the genitalia. For thepurpose of this specification, the mucosa will also include the externalsurface of the eye, i.e. the cornea.

The substance in combination with the Biovector may be added to anymucosal surface. Some particularly suitable mucosal surfaces include,for example, the nasal, buccal, oral, vaginal, ocular, auditory,pulmonary tract, urethral, digestive tract, or rectal surface.

The cross-linked polysaccharide or oligosaccharide preferably bindsnon-specifically to the mucosal surface. Applicants have unexpectedlydiscovered that non-specifically binding polysaccharides andoligosaccharides in accordance with the invention make superior carriersfor delivering substances to mucosal surfaces. This discovery issurprising since, as mentioned above, European Patent 352 295 of AccessPharmaceuticals reported the requirement for a multivalent binding agentspecific for endothelial surface determinants in carriers for drugs anddiagnostic agents.

Properties of Biovectors

The Biovector comprises a core of a natural hydrophilic polymer, suchas, for example, a cross-linked polysaccharide or a cross-linkedoligosaccharide, or a derivative or hydrolysate of a cross-linkedpolysaccharide or a cross-linked oligosaccharide, or a mixture thereof.The polysaccharide or oligosaccharide may be naturally cross-linked ormay be chemically cross-linked by methods known in the art. Somesuitable chemical cross-linking methods include, for example, contactingthe polysaccharide or oligosaccharide with a multi-functional agent,such as epichlorohydrin or phosphorous oxychloride. The minimum molarratio of cross-linking agent to glucose residue may be, for example,1:15, 1:12, or 1:10 in the case of phosphorous oxychloride and 1:50,1:40, or 1:30 in the case of epichlorohydrin. The maximum molar ratio ofcross-linking agent to glucose residue may be, for example, 1:0.5,1:0.7, or 1:1 in the case of phosphorous oxychloride and 1:2, 1:3, or1:5 in the case of epichlorohydrin. For epichlorohydrin, a preferredrange of ratios of cross-linking agent to glucose residue is 1:15 to1:7. For phosphorous oxychloride, a preferred range of ratios ofcross-linking agent to glucose residue is 1:7 to 1:2. When phosphorousoxychloride is used as the multi-functional agent, the cross-linkedproduct preferably comprises approximately 0.1 to 3.0 mmolephosphate/gram, preferably 0.4 to 1.0 mmole phosphate/gram, of finalproduct.

Some suitable examples of naturally cross-linked polysaccharidesinclude, for example, cellulose and its derivatives. Some suitableexamples of chemically cross-linked polysaccharides include, forexample, epichlorohydrin cross-linked starch, i.e. degradable starchmicrospheres (DSM), and epichlorohydrin cross-linked dextran, i.e.Sephadex.

The polysaccharides or oligosaccharides useful in the present inventionmay be derived from any saccharide monomer. Glucose is the preferredmonosaccharide. The polymers or oligomers may be formed from themonomers in either the α or β orientation, and may be linked at the 1-4or 1-6 positions of each saccharide unit. The polysaccharides oroligosaccharides preferably have a molecular weight between 1,000 to2,000,000 daltons, preferably 2,000 to 100,000 daltons, amd mostpreferably 3,000 to 10,000 daltons.

The preferred polysaccharides are starch (glucose α 1-4 polymers) anddextran (glucose α 1-6 polymers derived from bacteria). Starch isespecially preferred. Starch from any of the well known sources ofstarch is suitable. Some suitable sources of starch include, forexample, potato, wheat, corn, etc. Other suitable polysaccharidesinclude, for example, pectins, amylopectins, chitosan, andglycosaminoglycan.

The cross-linked polysaccharides or oligosaccharides may also bederivatives of hydrolysates of the cross-linked polysaccharides oroligosaccharides mentioned above. Some preferred hydrolysates of starchinclude, for example, acid hydrolyzed starch, such as dextrins, orenzyme hydrolyzed starch, such as maltodextrins. The hydrolysis degreeof the polysaccharide or oligosaccharide is determined by the reducingpower of the hydrolysate, commonly expressed as the Dextrose Equivalent(DE). The DE range preferably varies between 2 to 20, preferably 2 to12.

An ionic group (0 to 3 milliequivalents, preferably 0 to 2milliequivalents, of ionic charge per gram) is optionally grafted to thecross-linked polysaccharide or oligosaccharide. The ionic group may bean anionic group or a cationic group. The Biovectors preferably have aminimum of 0.2, 0.4, 0.6, or 0.8 milliequivalents of ionic charge pergram of polysaccharide core, and a maximum of 1.2, 1.4, 1.6, or 1.8milliequivalents of ionic charge per gram of polysaccharide core.Methods are known in the art for grafting ionic groups topolysaccharides and oligosaccharides.

The cross-linked polysaccharide or oligosaccharide may be made anionicby grafting a negatively charged or acidic group. Some suitable anionicgroups grafted to the polysaccharide or oligosaccharide include, forexample, phosphate, sulfate, or carboxylate. The anionic group may begrafted by treating the polysaccharide or oligosaccharide with anactivated derivative of a polyhydric acid, such as phosphoric acid,sulfuric acid, succinic acid, or citric acid. Activated derivatives ofpolyhydric acids include, for example, acyl halides, anhydrides, andactivated esters. The preferred anionic group is phosphate grafted viatreatment with phosphorous oxychloride. A Biovector to which a phosphategroup is grafted is referred to as SMBV-P.

The polysaccharide or oligosaccharide may be made cationic by grafting aligand that comprises a positively charged or basic group. Some suitablecationic groups grafted to the polysaccharide or oligosaccharideinclude, for example, quaternary ammonium ions, and primary, secondary,or tertiary amines. Some suitable ligands that can be grafted to thepolysaccharide or oligosaccharide include, for example, choline,2-hydroxypropyltrimethylammonium, 2-dimethylaminoethanol,2-diethylaminoethanol, 2-dimethylaminoethylamine, and2-diethylaminoethylamine. These ligands may be conveniently grafted tothe polysaccharide or oligosaccharide by methods known in the art, suchas, for example, by contacting the polysaccharide or oligosaccharidewith a suitable derivative of the respective alkyl group, such as achloride, bromide, iodide, or epoxide.

Another suitable method for grafting cationic groups to thepolysaccharide or oligosaccharide includes grafting a polyhydric acid,as described above, and then using a free acid group, such as a freecarboxylate group, to graft the basic ligand via, for example, an amideor ester bond. Amino acids are convenienly grafted this way. Somesuitable examples of amino acids include, for example, glycine, alanine,glutamic acid or aspartic acid.

The preferred cationic group is quaternary ammonium. A Biovector towhich a quaternary ammonium group is grafted is referred to as SMBV-Q.

It should be noted that Illum et al., International Journal ofPharmaceutics 39, 189-199 (1987), have reported finding no detectableamount of model drugs released from a cationic dextran microsphere,DEAE-Sephadex. Illum et al. attribute this lack of release to binding ofthe model drug to the cationic binding sites in the microsphere matrix.Applicants have, however, unexpectedly found efficient release ofsubstances from polysaccharides, to which cationic groups have beengrafted.

Optionally, the polysaccharide or oligosaccharide core of the Biovectoris covalently bonded to a layer of lipid compounds. The layer of lipidcompounds may coat the polysaccharide or oligosaccharide core eitherpartially or completely. The lipid layer preferably comprises naturalfatty acids, as described in International PCT Application WO 94/23701.

The cross-linked polysaccharide or oligosaccharide, either with orwithout a lipid layer, may also optionally be partially or completelycoated with one or more amphiphilic compounds. Such Biovectors arereferred to as light Biovectors or L-SMBV. Biovectors consisting only ofa core of cross-linked polysaccharide or oligosaccharide are referred toas core Biovectors.

The amphiphilic coating preferably adheres to the cross-linkedpolysaccharide or oligosaccharide, or to the optional lipid layer, bymeans of non-covalent bonds, such as by means of ionic or hydrogenbonds. The amphiphilic compounds suitable for the coating are selectedto confer a physico-chemical environment appropriate to the substance,the mode of mucosal administration, and the desired effect.

The amphiphilic coating may comprise any amphiphilic compound that canbe adsorbed on the surface of the core of the Biovector. Preferably, theamphiphilic coating comprises mainly a natural or synthetic phospholipidor ceramide, or a mixture thereof.

The phosphate group of the phospholipid may optionally be grafted toionic or neutral groups. Some suitable phospholipids include, forexample, phosphatidyl choline, phosphatidyl hydroxycholine, phosphatidylethanolamine, phosphatidyl serine, and phosphatidyl glycerol. Apreferred phospholipid is dipalmitoyl phosphatidylcholine (DPPC).

The amphiphilic coating may also comprise a derivative of a phospholipidor ceramide. Some suitable derivatives of phospholipids includePEG-phospholipids, and phospholipids grafted to other molecules orpolymers.

The amphiphilic coating may also comprise other amphiphilic compounds,either by themselves or in combination with the phospholipids,ceramides, or derivatives described above. Some suitable examples ofsuch other amphiphilic compounds include poloxamers, modifiedpolyoxyethylene, and other detergents and surface active compounds.

Additional compounds and mixtures thereof may be added to thephospholipids or ceramides in the amphiphilic coating. Some examples ofsuch additional compounds include fatty acids, steroids (such ascholesterol), triglycerides, lipoproteins, glycolipids, vitamins,detergents, and surface active agents.

The preparation of Biovectors may normally be conveniently carried out,either as a simple one-step process (in case of a core Biovector) or aas a two step process: the core is first prepared and then is coatedwith an amphiphilic compound to create a light Biovector.

The size of the Biovector is an important element of the presentinvention. For example, Ilium et al. have emphasized the importance ofmicrospheres having a size larger than 10 μm for nasal delivery. SeeIllum et al., International Journal of Pharmaceutics 39, 189-199 (1987).

Applicants have, however, unexpectedly found that Biovectors muchsmaller than 10 μm are highly efficient carriers for administeringsubstances to the nasal mucosa, as well as to other mucosa. TheBiovectors of the present invention preferably have a minimum diameterof about 20 nm, more preferably about 30 nm, and most preferably about40 nm. The maximum size of the Biovectors is about 200 nm, morepreferably about 150 nm, and most preferably about 100 nm. The optimalsize of the Biovector is between 60-90 nm, and most optimally about 80nm.

The relatively small size of the Biovectors confers various advantages,making the Biovectors even more suitable for administration to themucosa. For example, the Biovectors have larger relative surfaces andvolumes than larger nanospheres and microspheres. In addition, the smallsize of the Biovectors permit convenient sterilization bymicrofiltration, thereby avoiding the need for preservatives.

The Biovectors can be administered in various forms. For example, theBiovectors can be administered in dispersed form, such as suspensions orgels. The Biovectors can also be produced in dry form by methods knownin the art, and administered in a suitable metered-dosing device.

For example, a suspension or gel of dispersed Biovectors can be dried bylyophilization or spray drying. All light Biovectors, such as anionicand cationic light Biovectors, as well as all core Biovectors, such asanionic and cationic core Biovectors, can be dried. The Biovectors maybe administered in dry form, or may be resuspended (i.e. rehydrated) ina suitable medium, preferably a pharmaceutically acceptable aqueousliquid or gel, and administered. For the purposes of this application,resuspended Biovectors mean Biovectors that have been dried andresuspended in a suitable medium.

Substances for Mucosal Administration

The substance administered to a mammal in combination with a Biovectorin accordance with the present invention may be any substance that isadministered to a mammal. Some suitable substances include, for example,therapeutic agents, prophylactic agents, and diagnostic agents. Asubstance may be introduced for more than one purpose, such as, forexample, as combination therapeutic and prophylactic agents,prophylactic and diagnostic agents, and therapeutic and diagnosticagents.

The therapeutic agent may be any composition of matter used in thetreatment of diseases and conditions that afflict mammals. Some suitableexamples of therapeutic agents include a radiopharmaceutical, ananalgesic drug, an anesthetic agent, an anorectic agent, an anti-anemiaagent, an anti-asthma agent, an anti-diabetic agent, an antihistamine,an anti-inflammatory drug, an antibiotic drug, an antimuscarinic drug,an anti-neoplastic drug, an antiviral drug, a cardiovascular drug, acentral nervous system stimulator, a central nervous system depressant,an anti-depressant, an anti-epileptic, an anxyolitic agent, a hypnoticagent, a sedative, an anti-psychotic drug, a beta blocker, a hemostaticagent, a hormone, a vasodilator, a vasoconstrictor, a vitamin, etc.

The prophylactic agent that is administered to a mammal in combinationwith a Biovector according to the invention may be any prophylacticagent used for preventing or reducing the effect of any disease orcondition that afflicts mammals by any mechanism. For example, theprophylactic agent may be an antigen used in a vaccine against apathogen. The pathogen may, for example, be a virus or a microorganism,such as a bacterium, a yeast, or a fungus. The virus may, for example,be an influenza virus, such as Haemophilus influenzae; acytomegalovirus; HIV; a papilloma virus; a respiratory syncytial virus;a poliomyelitis virus; a pox virus, such as chicken pox virus (i.e.varicella zoster virus); a measles virus; an arbor virus; a Coxsackievirus; a herpes virus, such as herpes simplex virus; a hantavirus; ahepatitis virus, such as hepatitis A, B, C, D, E, or G virus; a lymedisease virus, such as Borrelia burgdorferi; a mumps virus, such asParamyxovirus; or a rotavirus, such as A, B, or C rotavirus.Particularly good results have been obtained with vaccines againstinfluenza virus and HIV.

A bacterium against which a vaccine according to the present inventionis effective may be any bacterium capable of causing disease in mammals.For example, the bacterium may be a member of the genus Neisseria, suchas N. gonorrhoeae and N. meningitidis; Aerobacter; Pseudomonas;Porphyromonas, such as P. gingivalis; Salmonella; Escherichia, such asE. coli; Pasteurella; Shigella; Bacillus; Helibacter, such as H. pylori;Corynebacterium, such as C. diphteriae; Clostridium, such as C. tetanii;Mycobacterium, such as M. etuberculosis and M. leprae; Yersinia, such asY. pestis; Staphylococcus; Bordetella, such as B. pertussis; Brucella,such as B. abortus; Vibrio, such as V. cholerae; and Streptococcus, suchas mutants Streptococci.

Other pathogens against which a vaccine according to the presentinvention is effective include, for example, a member of the genusPlasmodium, such as the species that causes malaria; a member of thegenus Schisostoma, such as the species that causes Schisostomiasis orBilharzia; and a member of the genus Candida, such as C. albicans.

The substance that can be combined with a Biovector may be a diagnosticagent. The diagnostic agent may be any composition of matter that isintroduced into a mammal for the purpose of detecting any disease orcondition, or to detect the concentration of a different substance addedto the mammal, such as a drug or a vaccine. For example, the diagnosticagent may be a contrast agent or an imaging agent, including a magneticimaging agent, that is capable of detecting an organ or other internalpart of the body of the mammal. Alternatively, the diagnostic agent maybe capable of detecting irregularities within the mammal, such asirregularities of the cornea, the respiratory tract, the digestivetract, the auditory canal, the urethra, the rectum, or any other part ofa mammal containing a mucosal membrane.

For the above purposes, the diagnostic agent is advantageously labeledwith a detectable group. The detectable group may, for example, be aradioactive group; a fluorescent group, such as, for example,fluorescene; a visible group, such as, for example, a marker dye; or amagnetic group, preferably suitable for magnetic resonance imaging.

The substance to be delivered in combination with a Biovector may, forexample, be a small chemical molecule or a biological molecule. A smallchemical molecule is usually a non-polymeric molecule that may or maynot occur naturally in the mammal to which it is administered. The smallchemical molecule may, for example, be an organic molecule, an inorganicmolecule, or an organo-metallic molecule. Some examples of smallchemical molecules include steroids, porphyrins, nucleotides,nucleosides, etc. as well as mixtures, and derivatives thereof.

Biovectors are particularly effective in delivering biological moleculesto the mucosa. For the purposes of this specification, a biologicalmolecule is a polymer of a type that occurs in nature, or a monomer ormoiety thereof. Such polymers typically comprise monomers such as aminoacids, nucleosides, nucleotides, and saccharides, and mixtures thereof.Some structural classes of biological molecules include, for example,amino acids, peptides, proteins, glycoproteins, and lipoproteins;proteoglycans; monosaccharides, oligosaccharides, polysaccharides, andlipopolysaccharides; fatty acids, including eicosanoids; lipids,including triglycerides, phospholipids, and glycolipids.

Additional biological molecules that can be delivered to the mucosa bymeans of Biovectors include nucleotides, nucleosides, and nucleic acidmolecules, including DNA and RNA polymers and oligomers. The nucleicacids may be, for example, ribozymes and antisense oligonucleotides.Nucleic acids may be administered for their own diagnostic ortherapeutic potential, or for their ability to be expressed inconnection with gene therapy.

Some functional classes of biological molecules include, for example,cytokines, growth factors, enzymes, antigens, (including epitopes ofantigens and haptens), antibodies, hormones (including both natural andsynthetic hormones and their derivatives), co-factors, receptors,enkephalins, endorphins, neurotransmitters, and nutrients. Some specificexamples of biological molecules include, for example, insulin, aninterferon, such as an α-, β-, or γ-interferon; an interleukin, such asany of IL-1 to IL15; any of the interleukin receptors, such as IL-1receptor; calcitonin; growth factors, such as erythropoietin,thrombopoietin, epidermal growth factor, and insulin-like growthfactor-1.

Administration of the substance in accordance with the present inventionmay be accompanied by one or more supplementary compound for enhancingthe activity, properties, or marketability of the substance. Forexample, adjuvants that enhance the absorption efficiency of the mucosaare known in the art. Some examples of such mucosa absorption enhancersinclude, for example, bile salts, such as sodium glycocholate, andsurfactants, such as polyoxyethylene-9-lauryl ether. Adjuvants forenhancing the immunogenicity of antigens are also known. Some examplesof immunogenicity enhancers include, for example, MPL, Quil A, QS 21,LPS, endotoxins, CTB, and BCG. Some additional supplementary compoundsinclude, for example, disinfectants, preservatives, surfactants,stabilizing agents, chelating agents, and coloring agents.

Another important feature of the present invention is the flexibility inadministering substances to the mucosa. For example, unlike most otherpharmaceutical carriers, the present invention provides for the deliveryof more than one substance per Biovector to be delivered to a mucosalsurface.

There is also flexibility in where the one, or more than one, substanceis located in the Biovector. For example, the one, or more than one,substance may be located in the inner core of the cross-linkedpolysaccharide or oligosaccharide. Alternatively, the one, or more thanone, substance may be located at the outer surface of the cross-linkedpolysaccharide or oligosaccharide.

If the cross-linked polysaccharide or oligosaccharide is coated with anamphiphilic layer, the one, or more than one, substance may be locatedin the inner core of the amphiphilic compound layer. Alternatively, theone, or more than one, substance may be located at the outer surface ofthe amphiphilic compound layer.

If more than one substance per Biovector is administered to a mammal,some or all of the substances may be located in the same part of theBiovector. Alternatively, some or all of the substances may be locatedin the different parts of the Biovector.

Methods are known for directing substances to various parts ofBiovectors. See International PCT Application WO094/20078.

As with other carriers, the substance may be pre-loaded in a Biovector,and the loaded Biovector stored prior to administration to the mammal.Preferably, however, the substance is post-loaded on an empty Biovectorjust prior to packaging or, such as in the case of labile substances forexample, the Biovector may be used as the dilution media for entrapingthe substance just prior to administration to the mammal. Methods areknown for pre-loading and post-loading Biovectors. See, for example,International PCT Applications WO 94/20078, WO 94/23701, and WO 96/06638of Biovector Therapeutics S.A.

Advantages of Mucosal Administration with Biovectors

Some of the advantages of the mucosal administration of substances tomammals may be seen by reference to the examples below. These advantagesare described for illustrative reasons only. The present invention isnot, however, in any way limited by the examples.

As shown in the experiment described in Example II, for example, theionic groups permit the mode of administration of Biovectors to bevaried according to the requirements of a particular case. The protocolis described in detail in Example II. Briefly, three cationicformulations and three anionic formulations of ¹⁴ C-labeled Biovectorswere administered intranasally to rats. At various times, the rats weresacrificed, and the percent of the label remaining in the nasal cavityand in the plasma was determined.

The results of this experiment, which are shown in FIG. 1, demonstratedthat approximately 30% of the dose of three cationic Biovectorsadministered intranasally to rats remained in the nasal cavity fiveminutes after administration, and was still present after twelve hours.

The good mucoadhesion of the cationic Biovectors increased the residencetime of the Biovector in the target mucosa. The increased residence timeis important where increased bioavailability or a local effect of theadministered substance is desired. A local effect of the administeredsubstance is desired under a variety of circumstances.

For example, a local effect is desired when an antibiotic or antiviraldrug is administered to treat a local bacterial or viral infection.Alternatively, a local effect is desired when a vaccine is administeredto protect a mamrnal against a mucosal infection by a microorganism orvirus. A third example of a situation where one desires a local effectis the administration of a diagnostic agent to image an organ thatcontains a mucosal membrane.

By contrast, the anionic Biovectors (SMBV-P1, SMBV-P2, and SMBV-P3),which exhibit comparable initial mucoadhesion (five minutes), have amore rapid clearance from the nasal mucosa than the cationic Biovectors.With the anionic Biovectors, less than 10% of the dose remaining fiveminutes after administration was found in the nasal cavities three hoursafter administration. There was no significant variation for the threeanionic formulations tested.

A significant amount of labeled anionic Biovectors was, however, foundin the plasma three hours and, to a lesser extent, six hours after nasaladministration of SMBV-P1, SMBV-P2, and SMBV-P3, respectively. SeeExample II and FIG. 2. Therefore, anionic Biovectors are of particularuse when a systemic response is desired.

In general, there are advantages in using positively charged Biovectorsfor administering Biovectors that have enhanced mucosal residency times.There are advantages in administering negatively charged Biovectors thathave enhanced ability to pass through the mucosa to the blood stream.The advantages of both charge types of Biovectors can be combined byadministering a mixture of a positively charged Biovector and anegatively charged Biovector.

The results of Example III confirm that in-vivo behavior ol anionicBiovectors (SMBV-P1, SMBV-P2, and SMBV-P3) is different from that ofcationic Biovectors (SMBV-Q1, SMBV-Q2, and SMBV-Q3). In this experiment,rats treated in accordance with the protocol of Example 2 weresacrificed after twelve hours, and the ¹⁴ C remaining in various organswas measured.

As expected, the relatively large amounts of ¹⁴ C from cationicBiovectors found in the nasal cavities, nasal cavity washings, andbronchi indicate an increased residence time of cationic Biovectors inthe mucosa in which, or near which, the Biovectors are administered. Forthe anionic Biovectors, the significant amount of ¹⁴ C found in theliver and kidney demonstrates the increased trans-mucosal passage of theBiovectors into the bloodstream.

The large amount of ¹⁴ C from both cationic and anionic Biovectors foundin the small and large intestine indicates that elimination ofBiovectors following nasal administration occurred through the digestivetract. The increase in the residence time of Biovectors in the digestivetract is especially significant for the oral administration of antigensassociated with Biovectors in the case of oral vaccination.

Further evidence for the good mucoadhesion of the cationic Biovectors isdemonstrated by the results shown in Example IV. In this experiment,fluorescein-labeled cationic light Biovectors as either dispersed orresuspended suspensions were administered intranasally to rats.Approximately 20% of the resuspended Biovectors adhere to the mucosaupon administration, and the same amount remains for at least twelvehours. The dispersed Biovectors do not adhere to the nasal mucosa afterthree hours, except at low levels. Approximately one third of theadministered fluorescent Biovectors are still found in suspension in thenasal washing five minutes after administration, but none is found sixhours later.

Example V provides important evidence of the superiority of Biovectorsin the mucosal administration of vaccines. In this experiment, acomparison was made between the intranasal (i.n.) administration of amonovalent split antigen of hemagglutinin (HA) and neuraminidase (N)prepared from viral membranes in cationic light Biovectors with theintranasal and subcutaneous (s.c.) administration of antigen alone. Theexperiment demonstrates that the antigen administered i.n. in aBiovector is able to elicit a superior mucosal and seric response.

Thus, the total IgG, specific IgG and inhibitory hemagglutination wereat the same order of magnitude when the antigen was administered i.n. ina Biovector compared to antigen administered s.c. alone. However, theantigen/Biovector formulation induces the production of circulating andsecretory IgA, while the antigen alone administered s.c. or i.n., forpractical purposes, did not.

Moreover, the ratio of specific IgG to total IgG in the nasal washingwas twice as high when the antigen was administered i.n. in a Biovectorthan when the antigen was administered alone s.c. A higher ratio meansthat the immune response is expected to be more specific and moreprotective. While not wishing to be bound by any theory, applicantsbelieve that membrane antigens such as those used in this experiment arepresented by the outer layer of the Biovector, creating a lipidsurrounding favorable for presenting the antigen to the immune system.

The experiment described in Example VI compares the effect of differentformulations of the gp160 protein of HIV on the mucosal immune responseof rabbits. The protein was administered with two formulations of apositively charged light Biovector, a dispersed formulation and anresuspended formulation. As a control, the protein was administered incombination with a potent mucosal adjuvant, subunit B of cholera toxin(CTB). In each of the three cases, a series of immunizations were madeat thirty day intervals. The first two immunizations were vaginal, thesecond two immunizations were oral, and the final immunization wasintramuscular.

The results showed that the Biovectors were at least as efficient as CTBin inducing specific IgA secretions in the vagina and in saliva ten daysafter the second vaginal administration, (D₄₀). The resuspended SMBVsinduced a 50% increase of the IgAs when compared to formulations of theantigen with CTB or in dispersed SMBV.

It should be noted that vaginal administration of the antigen inducedsecretion of specific IgAs in the saliva as well as in the vagina. Thus,the antigen, which entered the MALT (mucosal-associated lymphoid tissue)at the vaginal level, induced the secretion of IgAs in situ. Inaddition, the Biovector formulations were able to stimulate a robust IgAresponse in the saliva by entering the so-called "common mucosal immunesystem."

The experiment described in Example VII compares the intranasalimmunization of mice with influenza hemagglutinin in a controlformulation with that of four formulations of light Biovectors:dispersed and positively charged, dispersed and negatively charged,resuspended and positively charged, and resuspended and negativelycharged. The effect of pre-loading and post-loading each Biovectorformulation on the relative serum IgG titer after 28 days was measured.In addition, a comparison of the relative titer obtained byadministering the pre-loaded Biovectors to animals that were awake withthat obtained by administering the pre-loaded Biovectors to animals thatwere anesthetized was made.

As expected, the control subunit antigen without any carrier or adjuvantis not very immunogenic when administered intranasally to mice, eitheranesthetized or awake. Of the SMBV subgroups, the positively charged anddispersed Biovectors showed a significant improvement (by more than anorder of magnitude) of the titer over those obtained with the antigenalone or other Biovector formulations. Both the pre-loaded andpost-loaded Biovectors have generally comparable effects. Thisversatility of the Biovector can be of particular interest, allowingeither a mixing of the active substance with the Biovector uponadministration, or integration of the active substance with theBiovector prior to its use.

Surprisingly, the anesthetized animals did not show a significantincrease in antibody titers, suggesting that the deposition, if any, ofthe antigen in the lower respiratory tract or the lung had littlebiological effect.

EXAMPLES Example I

Preparation of Biovectors.

In the examples below, Biovectors, when labeled, are labeled before the, phospholipidation process. When loaded with one (or more than one)biologically active compound, the loading occurs after the process ofmanufacturing the empty Biovector.

I(a). Preparation of anionic core Biovector (SMBV-P1)

500 g of maltodextrine (Glucidex, Roquette, Lestrem, France) are pouredin a 10 liter reactor (TRIMIX) along with 2 liters of demineralizedwater. After solubilization at 4° C., 500 ml of sodium hydroxide (NaOH)10M are added with mechanical stirring. When the temperature of thesolution has stabilized at 4° C., 1700 ml of 10M NaOH and 283.3 ml ofPOCL₃ are added under controlled flow conditions. The cross linkingreaction takes place with mechanical stirring during a 20 hour period.At the end of the 20 hour period, the reacting mixture is stirred anadditional 15 minutes. A volume of 5 liters of demineralized water isadded and the pH is adjusted to 7.0 by neutralization with glacialacetic acid. The hydrogel obtained is ground under high-pressure. At theend of this step, the mean diameter of the particles is approximately 60nm. Further purification proceeds as follows: (i) microfiltration at0.451 μm to eliminate larger particles, (ii) diafiltration at constantvolume to eliminate smaller molecules (salts, fragments ofpolysaccharides, etc). The anionic polysaccharide cores (PSC) are thenconcentrated, added to sterile flasks, and stored at ˜20° C.

I(b). Preparation of dispersed anionic light Biovector (SMBV-P2)

Anionic core Biovectors are prepared as described in Example I(a), andlabeled as described when necessary. Thawed cores are diluted in osmosedwater in a glass flask at a concentration of 1 mg per milliliter (e.g.250 mg of PSC/250 ml of water). The dispersion is stirred 5 to 10minutes and homogenized in a high pressure homoginizer (RANNIE Lab) at400 bars for 3 minutes. The suspension is warmed at 80° C. in athermostated bath. The lipids of the future outer membrane (e.g. DPPC,DPPC/cholesterol, etc), in powder form, are added in a ratio of 0.3:1(w/w) of the PSC mass (e.g. 75 mg of lipids for 250 mg of PSC). Thelipids are mixed and solubilized in 2.5 ml of ethanol 95% (v/v). Thehomogenizer is warmed to 60° C. by closed water circulation. The ethanolsolution of lipids is injected in the suspension of PSC at 80° C. andthen homogenized at 450 bars for 25 minutes at 60° C. At the end of thisstep, the preparation is put in a glass container and free ethanol iseliminated from the light-Biovector preparation at reduced pressure. Theresulting light anionic Biovectors are filtered (0.2 μm) and stored.

I(c). Preparation of resuspended anionic light Biovector (SMBV-P3)

Anionic core and light Biovectors are suspended in water at aconcentration of 1.2 mg/ml, and then distributed in doses of 1 ml incryovials especially designed for freeze-drying. The cryovials areplaced on a freeze-dryer, (Dura dry, FT Systems), frozen at 30° C., andfreeze dried in stages, first -10° C., then 10° C., and finally 10° C.during the primary drying, and 30° C. for the following step. Drying isusually achieved in 24 hours. The lyophilized Biovectors in eachcryovial are rehydrated in 200 μm of PBS.

I(d). Preparation of cationic core Biovector (SMBV-Q1)

500 mg of maltodextrine (Glucidex, Roquette, Lestrem, France) aresolubilized with 0.880 liters of water at 20° C., with stirring, in athermoregulated reactor. Seven grams of NaBH₄ are added and mixed for 1hour. 220 ml of NaOH 10M are added, followed by 30.25 ml ofepichlorydrin (Fulka). After 12 hours of reaction, 382.3 g ofglycidyltrimethylammonium chloride (Fulka) are introduced and themixture is stirred for 10 hours. The resulting gel is diluted with 8liters of demineralized water and the pH is adjusted to 7.0 byneutralization with glacial acetic acid. The hydrogel obtained is groundunder high-pressure. The pressure used is 400 bars. At the end of thisstep, the mean diameter of the particles is approximately 60 nm. Furtherpurification proceeds as follows: (i) microfiltration at 0.45 μm toeliminate larger particles, (ii) diafiltration at constant volume toeliminate smaller molecules (salts, fragments of polysaccharides). Thecationic PSC are then concentrated, sampled in sterile flasks and storedat ˜20° C.

I(e). Preparation of dispersed cationic light Biovector (SMBV-Q2)

Cationic core Biovectors are prepared as described in Example I(d), andlabeled as described when necessary. Thawed cores are diluted in osmosedwater in a glass flask at a concentration of 1 mg per milliliter (e.g.250 mg of PSC/250 ml of water). The dispersion is stirred 5 to 10minutes and homogenized (RANNIE Lab) at 400 bars for 3 minutes. Thesuspension is warmed at 80° C. in a thermostated bath. The lipids of thefuture outer membrane (e.g. DPPC, DPPC/cholesterol, etc), in powderform, are added in a ratio of 0.3:1 (w/w) of the PSC mass (e.g. 75 mg oflipids for 250 mg of PSC). The lipids are mixed and solubilized in 2.5ml of ethanol 95% (v/v). A homogenizer is warmed to 60° C. by closedwater circulation. The ethanol solution of lipids is injected in thesuspension of PSC at 80° C. and then homogeneized at 450 bars during 25minutes at 60° C. At the end of this step, the preparation is put in aglass container and free ethanol is eliminated from the light Biovectorpreparation at reduced pressure. Light cationic Biovectors are filtered(0.2 μm) and stored.

I(f). Preparation of resuspended cationic light Biovector (SMBV-Q3)

Cationic core and light Biovectors are suspended in water at aconcentration of 1.2 mg/ml, and then distributed in doses of 1 ml incryovials especially designed for freezedrying. The cryovials are placedon a freeze-dryer, (Dura dry, FT Systems), frozen at -30° C., and freezedried in stages, first -10° C., then 0° C., and finally 10° C. duringthe primary drying, and 30° C. for the following step. Drying is usuallyachieved in 24 hours. The lyophilized Biovectors in each cryovial arerehydrated in 200 μl of PBS.

I(g). Labeling of Biovectors with ¹⁴ C cyanuric chloride

Polysaccharidic cores are labeled with radioactive ¹⁴ C triazine usingthe ability of cyanuric chloride to react with the free hydroxyl groupsof the polysaccharide. This reaction is carried out on the finishedpolysaccharide cores prepared as described above. The protocol below isrepresentative of any polysaccharide core (anionic or cationic).

¹⁴ C cyanuric chloride at 47 mCl/mmol is obtained following customsynthesis from Dupont NEN Product (Boston, Mass.). The cyanuric chlorideis suspended before use in pure acetonitrile at 100 g/l. Thepolysaccharide cores are suspended in water at 40 g/l, and the pH isadjusted to 10 with sodium carbonate. The suspension is warmed andmaintained at 50° C. The desired quantity of cyanuric chloride is added(normally between 1-5% w/w to polysaccharide cores) and the pH ismonitored with a pH-meter. The pH is maintained during the reaction byadding small portions of solid sodium carbonate and the reaction iscarried out during a period of five hours. Once the reaction iscompleted, the labeled polysaccharide core suspension is placed in aultrafiltration stirred cell equipped with a 10 Kdalton membrane(Amicon, France) and the solution is diafiltered against 1 mM pH 7.4phosphate buffer solution until no radioactivity is found in thefiltrate. The resulting suspension of labeled polysaccharide cores isthen sterilized by filtration through 0.2 μm filters and stored insterile containers. The radioactivity contents is determined bymeasurements on a Beckman Beta-Counter (Germany) and expressed in μCiper mg of polysaccharide cores. The resulting labeled polysaccharidecores can be used as described above to prepare labeled Biovectors.

1(h). Labeling of Biovectors with dichlorotriazinyl fluorescein.

Polysaccharide cores are labeled with dichlorotriazinylfluorescein usingthe ability of the dichlorotriazine moiety to react with the freehydroxyl groups of the polysaccharide. This reaction is carried out onthe finished polysaccharide cores prepared as described above. Theprotocol is representative of any polysaccharide core (anionic orcationic at any charge). Dichlorotriazinyl fluorescein is obtained fromSigma Chemicals (St. Louis, USA). The dichlorotriazinyl fluorescein issuspended before use in pure dimethyl formamide at 100 g/liter. Thepolysaccharide cores are suspended in a buffer solution (150 mM NaCl and140 mM sodium hydrogen carbonate) at 50 grams/liter and the pH isadjusted to 10 without sodium hydroxide. The desired quantity ofdichloro-triazinylfluorescein is added (normally between 1-5% w/w topolysaccharide cores) and the reaction is allowed to stand five hours atroom temperature with gentle stirring. Once the reaction is finished,the labeled polysaccharide core suspension is placed in anultrafiltration stirred cell equipped with a 30 kdalton membrane(Amicon, France), and the solution is diafiltered against water until nofluorescence is found in the filtrate. The resulting labeledpolysaccharide core suspension is then sterilized by filtration on 0.2μm filters and conditioned on sterile containers. The fluorescencecontent is determined by measurements on a Perkin Elmer LuminescenceSpectrophotometer LS 50 B. The resulting polysaccharide cores can benormally used after labeling to prepare SMBV suspensions as describedabove.

I(i). Large scale preparation of dispersed light Biovectors

Modifications may be made to the proceedures described in Examples I(b)and I(e) to assist in scaling up the procedures. The duration times ofthe high pressure homoginization steps are varied on the basis of thevolume and the concentration of light Biovectors to be prepared. Thesecond high pressure homoginization step may be eliminated, and replacedby incubation of the light Biovectors at 80° C. with stirring. Theelimination of ethanol may be accomplished by means of diafiltrationagainst water rather than at reduced pressure.

Example II

Adhesion of ¹⁴ C-Labeled Biovector on Nasal Mucosa of Rats.

Male Sprague Dawley rats of approximately 200 g each were divided intosix groups according to the type of labeled Biovector administered. Thesix types of Biovector are summarized in Table II-1 below:

                                      TABLE II-1                                  __________________________________________________________________________    Characteristics of Biovectors used for intranasal pharmacokinetic and         biodistribution studies. PSC Charge refers to the milliequivalents of         ionic charge per                                                              gram of polysaccharide core. ND means not determined.                         Samples                                                                             SMBV-P1                                                                             SMBV-P2                                                                             SMBV-P3                                                                             SMBV-Q1                                                                             SMBV-Q2                                                                             SMBV-Q3                                   __________________________________________________________________________    Type  Core  Light Light Core  Light Light                                     Example                                                                             I(a)  I(b)  I(c)  I(d)  I(e)  I(f)                                      Charge type                                                                         Anionic                                                                             Anionic                                                                             Anionic                                                                             Cationic                                                                            Cationic                                                                            Cationic                                  PSC Charge                                                                          1.79 mEq/g                                                                          1.79 mEq/g                                                                          1.79 mEq/g                                                                          1.85 mEq/g                                                                          1.85 mEq/g                                                                          1.85 mEq/g                                PSC mean                                                                            55 nm 55 nm ND    68 nm 68 nm ND                                        diameter                                                                      State Dispersed                                                                           Dispersed                                                                           Resuspended                                                                         Dispersed                                                                           Dispersed                                                                           Resuspended                               __________________________________________________________________________

Each rat in groups SMBV-P1 and SMBV-Q1 received a dose of 100 μg of itsrespective ¹⁴ C-labeled Biovector formulation administered withoutanesthetic intranasally in a volume of 50 μl of suspension (25 μl ineach nostril).

Each rat in groups SMBV-P2, SMBV-P3, SMBV-Q2, and SMBV-Q3 received adose of 150 μg of its respective ¹⁴ C-labeled Biovector formulationadministered without anesthetic intranasally in a volume of 50 μl ofsuspension (25 μl in each nostril).

The above doses represent approximately 200 μl of suspension ofBiovectors per kg of rat. This volume of suspension is equivalent toapproximately 400 μg of polysaccharide and approximately 200 μg of lipidper kg of rat.

At 0.083 hours (five minutes), three hours, six hours, twelve hours, andtwenty four hours, three rats in each group were sacrificed. Both nasalcavities were isolated; the nasal tract was opened and washed with 5 mlof physiological saline; and blood was taken and centrifuged. The ¹⁴ Cremaining in the nasal washing, nasal cavity, and plasma were measured.The results are shown in FIGS. A and B.

Example III

Biodistribution of ¹⁴ C-Labeled Biovector After Nasal Administration.

Male Sprague Dawley rats of approximately 200 g each were treated asdescribed in Example II. Twelve hours after nasal administration, threerats per sample were sacrificed, and the ¹⁴ C remaining in the liver,spleen, kidney, blood, bronchi, lung, oesophagus, stomach, small andlarge intestine, skeletal muscle, sub-maxillary lymph node, brain, andnasal turbinate was measured.

Table III-1 below summarizes the biodistribution twelve hours afternasal administration of the Biovector formulations described in TableII-1.

                                      TABLE III-1                                 __________________________________________________________________________    Twelve hours biodistribution after nasal administration of different          Biovector                                                                     formulations. The Biovectors are described in Table II-1 above. Blq =         below level of quantification.                                                Organs                                                                              SMBV-P1                                                                              SMBV-P2                                                                              SMBV-P3                                                                              SMBV-Q1                                                                             SMBV-Q2                                                                             SMBV-Q3                                __________________________________________________________________________    Bronchi                                                                             blg    blg    blg    1.05 ± 1.81                                                                      blq   1.34 ± 1.53                         Lung  blq    blq    blq    blq   blq   0.15 ± 0.11                         Oesophagus                                                                          0.05  ± 0.04                                                                      blq    blq    blq   blq   blq                                    Stomach                                                                             0.23 ± 0.08                                                                       0.44 ± 0.48                                                                       0.38 ± 0.41                                                                       0.55 ± 0.18                                                                      1.56 ± 0.98                                                                      3.20 ± 4.60                         S-L   46.9 ± 8.1                                                                        48.6 ± 16.7                                                                       43.0 ± 19.5                                                                       42.0 ± 8.7                                                                       52.3 ± 21.5                                                                      33.4 ± 16.1                         Intestine                                                                     Spleen                                                                              blq    blq    blq    blq   blq   blq                                    Liver 0.72 ± 0.49                                                                       0.46 ± 0.03                                                                       0.33 ± 0.06                                                                       blq   blq   blq                                    Kidney                                                                              0.31 ± 0.16                                                                       0.34 ± 0.14                                                                       0.23 ± 0.02                                                                       0.04 ± 0.01                                                                      0.04 ± 0.01                                                                      blq                                    Brain blq    blq    blq    blq   blq   blq                                    Muscle                                                                              blq    blq    blq    blq   blq   blq                                    Lymph blq    blq    blq    blq   blq   blq                                    Node                                                                          Plasma                                                                              blq    blq    blq    blq   blq   blq                                    Turbinate                                                                           0.54 ± 0.64                                                                       0.68 ± 0.90                                                                       0.51 ± 0.54                                                                       11.4 ± 7.0                                                                       8.7 ± 8.8                                                                        8.6 ± 7.4                           Washing                                                                             0.007 ± 0.002                                                                     0.008 ± 0.003                                                                     0.008 ± 0.002                                                                     0.64 ± 1.03                                                                      0.25 ± 0.20                                                                      0.26 ± 0.38                         Fluid                                                                         __________________________________________________________________________

Example IV

Adhesion of Fluorescent Biovectors to the Nasal Mucosa of Rats.

Three groups of anesthetized Males Wistar rats (twelve rats in eachgroup) received a single drop in the nostrils of 50 μl of either asuspension of PBS/glycerol (control); a suspension of 0.93 mg/ml offluorescein-labeled cationic light Biovectors (Example I(h)) suspendedin PBS/glycerol (dispersed L-SMBV); or a suspension of 0.93 mg/ml oflyophilized cationic fluorescein-labeled light Biovectors re-suspendedin PBS/glycerol (resuspended L-SMBV). The dispersed SMBV have diametersof approximately 80 nm, and polysaccharide cores grafted with quaternaryammonium ions.

At times zero, five minutes, three hours, six hours, and twelve hours,two rats from each group were sacrificed. The fluorescence was measuredfrom both the nasal washings (three washings with NaCl) and in the nasalmucosa (scratching). The results are shown in the Tables IV-1 and IV-2below.

                  TABLE IV-1                                                      ______________________________________                                        Percent of fluorescence remaining in the nasal washings.                      NASAL                                                                         WASHINGS     5 min.  3 hr.     6 hr.                                                                              12 hr.                                    ______________________________________                                        disp. SMBV-Q 28%     3%        0%   0%                                        res. SMBV-Q  30%     1%        0%   0%                                        ______________________________________                                    

                  TABLE IV-2                                                      ______________________________________                                        Percent of fluorescence remaining in the nasal cavity.                        NASAL                                                                         MUCOSA       5 min.  3 hr.     6 hr.                                                                              12 hr.                                    ______________________________________                                        disp. SMBV-Q 40%      4%        3%   0%                                       res. SMBV-Q  21%     20%       20%  20%                                       ______________________________________                                    

Histological studies conducted in parallel showed that the observedfluorescence is not granular, and is generally visible at the apicalpole of the cells.

Example V

Comparison of Intranasal Administration of a Monovalent Split of anInfluenza Virus Antigen in Biovectors With Intranasal (i.n.) andSubcutaneous (s.c.) Administrations of HA Alone.

The antigen used in this study was a commercially available monovalentsplit of hemagglutinin (HA) and neuraminidase (N) prepared from viralmembranes. The study was performed by administering 5 μg of the antigenin three groups of six BALB/c mice per group. Two groups received alone,one group i.n. and the other group s.c. The third group received antigenin a dispersed, positively charged Biovector (SMBV-Q) that had anamphiphilic layer (DPPC/chol in a ratio of 70:30) prepared in accordancewith Example I(e). The antigen was injected subcutaneously (s.c.) oradministered intranasally (i.n.) at day zero and day twenty one. Theantibody response was analyzed at day thirty five by ELISA and byinhibitory hemagglutination against Nibl6. The results are shown inTable V-1:

                  TABLE V-1                                                       ______________________________________                                        Response to administration i.n. of antigen.                                                             ELISA responses in nasal                            Administration            pharyngeal washings                                 of       Total ELISA responses in sera                                                                  Specific  Specific                                  Flu Vaccine                                                                            IgG      IHA     IgA   IgG     IgA                                   ______________________________________                                        subcutaneous                                                                           350 000  320     0     64       0                                    intranasal                                                                              2 000   0       0     0        1,5                                  intranasal in                                                                          145 000  240     581   48      128                                   Disp. SMBV-Q                                                                  ______________________________________                                    

Example VI

Comparison of Routes of Administration of gp16O of HIV With Biovectors.

Several successive immunizations at one month intervals were made inrabbits against the protein gp160 of HIV: two vaginal applications, twooral administrations and one intramuscular injection. Four femalerabbits received five doses of 10 μg of gp160 of HIV, formulated ineither:

(a) a solution containing the subunit B of the cholera toxin (CTB), theexotoxin of Vibrio cholerae, which is a potent mucosal adjuvant.

(b) a solution of positively charged, dispersed light Biovectors (disp.SMBV-Q)

(c) a solution of lyophilized, positively charged light Biovectorsresuspended in PBS (resuspended light Biovectors--res. SMBV-Q).

Immunizations were made as follows: vaginal at day D₀ and D₃₀ , oral atday D₆₀ and D₉₀ and intramuscular at day D₁₂₀.

Ten days after each immunization (days D₄₀, D₇₀, D₁₀₀ and D₁₃₀), thespecific IgAs in the vagina mucosa and in the saliva were measured byELISA. The results are shown in the Table below.

                  TABLE VI-1                                                      ______________________________________                                        Vaginal administration of gp160 of HIV delivered by Biovectors                           IgAs in vagina at D.sub.40                                                                IgAs in saliva at D.sub.40                             ______________________________________                                        gp160-CTB    0.41          0.42                                               gp160-disp SMBV-Q                                                                          0.42          0.42                                               gp160-res. SMBV-Q                                                                          0.65          0.60                                               ______________________________________                                    

                  TABLE VI-2                                                      ______________________________________                                        Oral Administration of gp 160 of HIV delivered by Biovectors                               IgAs in saliva                                                                         IgAs in vagina                                                       D.sub.70                                                                            D.sub.100                                                                            D.sub.100                                           ______________________________________                                        gp160-CTB      0.42    0.38   0.28                                            gp160-disp SMBV-Q                                                                            0.47    0.35   0.29                                            ______________________________________                                    

Table VI-2 shows that, after the last vaginal administration, oraladministration maintains the mucosal immunity at the same level.

Again, the Biovectors are as least efficient as CTB in maintainingspecific IgA secretion by the vagina and the saliva.

                  TABLE VI-3                                                      ______________________________________                                        Intramuscular Administration of gp160 of HIV Delivered by Biovectors          Day D.sub.130  IgAs in saliva                                                                           IgAs in vagina                                      ______________________________________                                        gp160-CTB      0.16       0.08                                                gp160-disp SMBV-Q                                                                            0.05       0.16                                                ______________________________________                                    

Table VI-3 shows that, at day D₁₃₀, the mucosal immunization does notpersist. The intramuscular injection is not able to re-boost it.

The Biovector therefore appears to induce mucosal immunity when used todeliver antigens at the mucosal level. It is a vector of activecompounds particularly adapted to mucosal administrations.

Example VII

Influenza Hemagglutinin Delivered Intranasally by Biovectors Samples offour female mice were immunized at day D₀ and boosted at D₁₄ with 5 μgof hemagglutinin (HA) applied intranasally in 20 μl or 50 μl of a PBSsolution or suspension, either alone or in a Biovector forrnulation. Onegroup of animals was subjected to light ether anesthesia (*), while theothers were awake. Administration of 20 μl on the outer nostrils ofawake animals restricts the antigen to the upper respiratory tract. Avolume of 50 μl directly into the nostrils of anesthetized animalsresults in deposition of at least part of the antigen in the lowerrespiratory tract and the lung, besides deposition in the nasal cavity.

Four different Biovectors were used: positively (SMBV-Q) and negatively(SMBV-P) charged light Biovectors, either resuspended (res) or dispersed(disp).

The influenza virus subunit antigen was either pre-loaded in theBiovectors (HA in SMBV) or simply post-loaded (HA+SMBV), i.e., admixedwith them immediately before administration to the animals. The antigenalone was used as a control. The quality of the material used in thepresent study was equivalent to that for human vaccination purposes.

At day D₂₈, the mice were sacrificed. Serum samples were taken from thevena porta and antigen specific antibodies were measured in a directenzyme-linked immunosorbent assay (ELISA). The results are shown inTable VII-1.

                  TABLE VII-1                                                     ______________________________________                                        Response to i.n. administration of HA in Biovectors. The numbers are          serum IgG titers (geometric mean) determined as the reciprocal of the         sample dilution that corresponds to an absorbance at 492 nm of 0.2            above background.                                                                                      anesthetized                                                 awake animals    animals                                              Administration   HA in                   HA in                                of HA     HA     SMBV     HA + SMBV                                                                              HA    SMBV                                 ______________________________________                                        alone     100                      200                                        disp SMBV-P      200      200            60                                   res SMBV-P       400      30             30                                   res SMBV-Q       10       20             30                                   disp SMBV-Q      2000     2000           4000                                 ______________________________________                                    

We claim:
 1. A method for the mucosal administration of a vaccineagainst a pathogen to a mammal, the method comprising contacting amucosal surface of the mammal with an antigen in combination with aBiovector core, wherein the Biovector core comprises a natural polymer,or a derivative or a hydrolysate of a natural polymer, or a mixturethereof, and wherein the core is uncoated, or is partially or completelycoated with no more than one layer, the layer comprising a lipidcompound covalently bonded to the core, or an amphiphilic compound. 2.The method of claim 1, wherein the natural polymer is selected from thegroup consisting of a cross-linked polysaccharide, a cross-linkedoligosaccharide, a derivative or hydrolysate of a cross-linkedpolysaccharidec or a cross-linked oligosaccharide, and a mixturethereof.
 3. The method of claim 2, wherein the cross-linkedpolysaccharide and cross-linked oligosaccharide are selected from thegroup consisting of starch, dextran, dextrin, and maltodextrin.
 4. Themethod of claim 2, wherein 0 to 2 milliequivalents of ionic charge pergram is grafted to the cross-linked polysaccharide or cross-linkedoligosaccharide.
 5. The method of claim 4, wherein the ionic charge is apositive charge.
 6. The method of claim 5, wherein the positive chargeis due to the presence of a cationic or basic group selected from thegroup consisting of a quaternary ammonium group, a primary amine, asecondary amine, and a tertiary amine.
 7. The method of claim 5, whereinthe positive charge is due to the presence of a quaternary ammoniumgroup.
 8. The method of claim 5, wherein the positive charge is due tothe presence of a ligand selected from the group consisting of choline,2-hydroxypropyltrimethylammonium, 2-dimethylaminoethanol,2-diethylarninoethanol, 2-dimethylaminoethylamine, and2-diethylaminoethylamine and an amino acid.
 9. The method of claim 4,wherein the ionic charge is a negative charge.
 10. The method of claim9, wherein the negative charge is due to the presence of a an anionic oracidic group selected from phosphate, a sulfate, and carboxylate. 11.The method of claim 9, wherein the negative charge is due to thepresence of a phosphate group.
 12. The method of claim 2, wherein thecross-linked polysaccharide or cross-linked oligosaccharide is coatedpartially or completely with a layer of an amphiphilic compound.
 13. Themethod of claim 12 wherein the amphiphilic compound is a phospholipid ora ceramide.
 14. The method of claim 13 wherein the phospholipid isselectcd from the group consisting of phosphatidyl choline, phosphatidylhydroxycholine, phosphatidyl ethanolamine, phosphatidyl serine, andphosphatidyl glycerol.
 15. The method of claim 1, wherein the diameterof the Biovector is 20-200 nm.
 16. The method of claim 1, wherein thediameter of the Biovector is 20-100 nm.
 17. The method of claim 1,wherein the cross-linked polysaccharide or cross-linked oligosaccharidebinds non-specifically to the mucosal surface.
 18. The method of claim 1wherein the Biovector is dispersed.
 19. The method of claim 1, whereinthe Biovector is dried.
 20. The method of claim 19, wherein the driedBiovector is resuspended.
 21. The method of claim 1, wherein thepathogen is selected from the group consisting of a virus, a bacterium,a yeast, and a fungus.
 22. The method of claim 21, wherein the virus isselected from the group consisting of an influenza virus, acytomegalovirus, HIV, a papilloma virus, a respiratory syncytial virus,a poliomyelitis virus, a pox virus, a measles virus, an arbor virus, aCoxsackie virus, a herpes virus, a hantavirus, a hepatitis virus, a lymedisease virus, a mumps virus, and a rotavirus.
 23. The method of claim22, wherein the virus is an influenza virus.
 24. The method of claim 22,wherein the virus is HIV.
 25. The method of claim 21, wherein thebacterium is selected from the group consisting of a member of the genusNeisseria, Aerobacter, Pseudomonas, Porphyromonas, Salmonella,Escherichia, Pasteurella, Shigella, Bacillus, Helibacter,Corynebacterium, Clostridium, Mycobacterium, Yersinia, Staphylococcus;Bordetelia, Brucelia, Vibrio, and Streptococcus.
 26. The method of claim21, wherein the pathogen is a member of a genus selected from the groupconsisting of Plasmodium, Schisostoma, and Candida.
 27. The method ofclaim 1, wherein the antigen is a biological molecule.
 28. The method ofclaim 27, wherein the biological molecule is selected from the groupconsisting of an amino acid, an oligopeptide, a peptide, a protein, aglycoprotein, and a lipoprotein.
 29. The method of claim 1, wherein morethan one antigen is administered in combination with the Biovector. 30.The method of claim 2, wherein the antigen is located in the inner coreof the cross-linked polysaccharide or cross-linked oligosaccharide. 31.The method of claim 2, wherein the antigen is located at the outersurface of the cross-linked polysaccharide or cross-linkedoligosaccharide.
 32. The method of claim 12, wherein the antigen islocated in the inner core of the amphiphilic compound layer.
 33. Themethod of claim 12, wherein the antigen is located at the outer surfaceof the layer.
 34. The method of claim 1, wherein the antigen is added tothe Biovector prior to administration to the mammal.
 35. The method ofclaim 1, wherein the antigen and the Biovector are mixed together at thetime of administration to the mammal.
 36. The method of claim 1, whereinthe mucosal surface is selected from the group consisting of a nasal,buccal, oral, vaginal, ocular, auditory, pulmonary tract, urethral,digestive tract, and rectal surface.
 37. The method of claim 36, whereinthe mucosal surface is selected from the group consisting of a nasal,vaginal, and ocular surface.